Human alcohol dehydrogenase (ADH, EC 1.1.1.1) is the primary enzyme responsible for ethanol metabolism in humans. ADH exists as 3 classes, Class I, II, III, that can be isolated from liver and have been differentiated on the basis of their electrophoretic (Vallee et al., 1983, Isozymes: Current Topics in Biological & Medical Research pp. 219-244, Liss, New York), immunological (Montavon et al., 1989, Anal Biochem 176 48-56), catalytic (Wagner, et al, 1983, Biochemistry 22: 1857-1863; Wagner et al., 1984. Biochemistry 23: 2193-2199; Ditlow et al., 1984, Biochemistry 23: 6363-6368), and structural (Strydom et al., 1982, Anal. Biochem. 123: 422-429; Kaiser et al, 1988, Biochemistry 27: 1132-1140) differences. The ADH classes further contain isozymes, particularly Class I ADH. Class I isozymes include homodimers and heterodimers composed of .alpha., .beta. and .gamma. subunits, for example, .alpha..beta..sub.1, .alpha..gamma..sub.1, .beta..sub.1 .beta..sub.1, .beta..sub.1.gamma.1, .beta..sub.1.gamma.2 and .sub..gamma.1.gamma.1. The occurrence of particular isozymes in some tissues is remarkably selective For example, Class III (.chi.) ADH is the only isozyme in brain and placenta and virtually the only isozyme in testis, while Class II (.eta.) has only been observed in liver homogenates. Vallee et al., 1983, supra. Since liver ADH isozymes are clearly responsible for the oxidation of most of the ethanol ingested, their involvement in the physiological and pathological consequences of human ethanol consumption is of considerable interest.
The classes of ADH, as well as individual isozymes differ in their substrate and inhibitor specificities. Wagner et al , 1983, Biochemistry 22: 1857-63; Deetz et al., 1984, Biochemistry 23 6822-28; Ditlow et al., 1984, Biochemistry 23: 6863-68; Mardh et al., 1985, Proc. Natl. Acad. Sci. USA 83: 2836-40. For example differences in activity toward a number of aromatic alcohols and aldehydes in norepinephrine (Mardh et al., 1986, Proc. Natl. Acad Sci. USA 83: 8908-12; Mardh et al., 1985, Proc. Natl. Acad. Sci. USA, 82: 4979-4982), dopamine (Mardh et al., 1986, Biochemistry, 25: 7279-7282) and serotonin metabolism (Consalvi et al., 1986, Biochem. Biophys. Res. Commun., 139: 1009-1016) have been observed. However, these or other substrates capable of measuring ADH activity cannot differentiate among classes, isozymes, or phenotypic ADH variants of isozymes. For this reason, investigations of the function of human ADH have long been hindered by the lack of sensitive and specific assays to detect the activity of and characterize the individual forms of ADH in human body fluids and tissues. The capacity to accomplish this would advance studies of their distribution and regulation and might help to elucidate genetic factors underlying alcohol use and abuse. Vallee, 1966, Therap. Notes 73: 71-4. There is increasing evidence of a genetic predisposition to certain forms of chronic alcoholism (Bohman. 1978, Arch. Gen. Psychiat. 35: 269-276).
Little is known at present regarding the genetics and distribution of the three ADH classes let alone the manner in which any differences between them might be manifested metabolically. It would clearly be advantageous if their existence, preponderance and variability could be ascertained in vivo from accessible tissues and body fluids The detection of ADH activity in such tissues or fluids has proven exceedingly difficult.
Total ADH activity in serum has been measured previously using a variety of substrates and methods but none of them have been specific or selective for isozymes or isozyme classes. Purified human isozymes or isozyme classes in fact have not been available for calibration or control and efforts to achieve indirect differentiation of Class II from Class I activity in serum and tissues based on differential pyrazole inactivation have only been inferential. Skursky et al., 1980, Drug and Alcohol Dependence, 6: 187-190.
Several assays for ADH activity have been developed to study its distribution in tissues and body fluids with the objective of determining possible prognostic and/or diagnostic characteristics Skursky et al., 1979, Anal. Biochem., 99: 65-71; Agarwal et al, 1982, Fresenius Anal. Chem. 311: 314., Kato et al., 1984, Clin Chem , 30: 1817-1820. Using conventional assay methods based on NAD reduction ADH activity is virtually undetectable in the serum of normal individuals. Thus, efforts to employ serum ADH for differential diagnostic purposes have been quite disappointing and conclusions regarding the usefulness of increased ADH activity to assess common types of liver disease remain in question. Khayrollah et al., 1982 Anal. Clin. Biochem., 19: 35-42. In addition any ADH activity measurements thus far reported are measurements of undifferentiated ADH activity, i.e., the sum of all activities of isozymes present Conventional methods, therefore, lack sufficient sensitivity to routinely detect ADH activity in normal sera and sufficient specificity or selectivity to measure the activities of different ADH isozyme classes in serum or other body fluids and tissues. The present invention solves these problems by providing for the first time, assay methods that are both sensitive enough to detect levels of ADH activity in sera and other body fluids and tissues, and, more importantly, selective enough to differentiate Class I from Class II ADH activity in such body fluids or tissues. The present invention also provides for the first time, highly purified human ADH isozymes and isozyme classes as standards for calibration and controls of enzyme activity in the novel assay methods of the present invention. In particular, highly purified cofactor-free Class I isozymes are recovered in greater yield and with higher specific activities as compared to corresponding isozymes prepared by conventional methods.